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The Importance of Negative/Counter Selection


In order to remove non-specific binding aptamers, a negative/counter selection step may be incorporated into the selection design. Undesired sequences can often survive the in vitro selection process through various mechanisms and if left unchecked, these sequences can be selectively amplified over the sequence of interest. Although the terms negative and counter selection have been used interchangeably, negative selection often refers to the removal of sequences that bind ligands found within the experimental setup. For example, removal of sequences that non-specifically bind to the microcentrifuge tube or the solid-support where the target molecule is immobilized is a form of negative selection. 

A counter selection is defined by the removal of sequences that bind to molecules similar to your intended target. For example, in vitro selection experiment screening for aptamers against theophyllin may consider incorporating a counter selection step against the xanthine family of molecules as they are structurally similar (e.g caffeine), thereby increasing the specificity of the aptamer.     

Although this technique certainly aids in directing the selection process, a single negative/counter selection step will not remove all unwanted sequences. There is a whole host of other challenges that need to be considered and if their removal is possible through your experimental design, then it is highly encouraged that it is implemented. In every in vitro selection experiment, the objective is to selectively enrich for your desired aptamer, while suppressing the growth of undesired sequences. Unfortunately, at the start of your experiment, there will inherently be a significant number of undesired sequences that will outcompete and possibly inhibit your desired aptamer sequences. If these sequences are not controlled, either a false positive will be observed at the end of your experiment or the sequences was not identified during sequencing. 

In the simplest example, panel A in the figure below depicts only two sequences (of equal activity) in a ratio of 2:1 for Undesired:Desired sequences. During sequencing, this ratio will remain the same but only 1/3 of all sequencing data will be useful. As a result, a typical sequencing experiment will submit ~90 clones and only 30 of that total can be used to identify aptamer enrichment. However, this is never the case in a real in vitro selection experiment as the number of non-specific aptamers vastly outnumbers the desired sequences. In panel B, if there are even just 1000 copies at the start, the probably of locating even 1 copy of the aptamer is extremely low.